The eyeless (ey), eyes absent (eya), and sine oculis (so) genes in Drosophila are key components of the retinal determination (RD) network, which is essential for normal development in both flies and humans. Eya functions both as a transcriptional coactivator and protein phosphatase while ey and so encode DNA-binding transcription factors. All three genes are necessary and sufficient for retinal development in Drosophila, as is the ey paralog twin of eyeless (toy). Moreover, the RD proteins physically interact and act synergistically in highly potent transcription complexes that regulate development of several organ systems. Two highly conserved homologs of so, Six1 and Six2, are required for normal retinal development in vertebrates as is the ey homolog Pax6. In humans, mutations in EYA1 and SIX1 cause the autosomal dominant disorder known as BOR (branchio-oto-renal) syndrome, characterized by branchial arch abnormalities, hearing loss, and kidney defects while loss of PAX6 causes aniridia. Despite their importance during mammalian development, the mechanism of Ey, Toy, Eya and So action remains incompletely understood. So directly activates ey prior to overt retinal differentiation, and is then required, along with ea, to repress ey transcription during photoreceptor differentiation. Thus, Eya and So mediate the transition from determination to differentiation and thereby act at a critical junction in organogenesis. Our proposal focuses on understanding the role of these genes in a well-characterized genetic system, the Drosophila eye. We will use a combination of genetics, molecular biology, and biochemistry to analyze the roles of Ey, Toy, Eya, and So during retinal development. Since genetic pathways are often conserved and reiteratively used during organ formation across phylogeny, studying the development of simpler organisms can provide rapid and significant insight into human disease.